X-ray crystallography is a technique that is widely used to obtain information about the structure of molecules. The technique exploits the characteristic diffraction of X-rays from crystals such that three-dimensional models may be generated of the constituent molecules of the crystal, which are consistent with the observed diffraction.
X-ray crystallography is often used in the context of biological research to establish the three dimensional structure of molecules of interest, and, in particular, that of proteins. In this context, X-ray crystallography is often referred to as protein crystallography. Recently there has been a significant international investment in the techniques of protein crystallography including protein production, purification, crystallisation, X-ray diffraction, data reduction and structure solution leading to “high-throughput” approaches. Despite this the technique often remains cumbersome with a high attrition rate in getting from expressed protein to structure.
A particularly laborious bottleneck is the crystallisation process by which the generation of single crystals suitable for X-ray crystallography from sparse quantities of protein in a liquid medium is attempted. A typical method for crystallisation of proteins is vapour diffusion, for example the “hanging drop” or “sitting drop” methods, whereby microliter or nanoliter drops of a protein and crystallisation reagent in solution are introduced to a well containing a reservoir of crystallisation reagent in solution at a higher concentration. There is only a short distance between the drop and the reservoir solution in the same well. The well is then sealed and due to different concentrations of the droplet and reservoir solution, water diffuses out of the droplet and protein crystals may form. Other crystallisation techniques include the microbatch technique, the dialysis technique, and the gel crystal growth method.
Typically single crystals are grown in containers with a plurality of wells, with each well containing droplets of different crystallisation solutions and/or different proteins. Frequently, in instances where the optimal crystallisation conditions are not known, the multi-well containers may be set up to screen a range of crystallisation conditions with, for example, a range of concentrations of crystallisation solutions, or pH, or other parameters. Crystalline material may form in some of the wells over a period of days or weeks. In such cases it is necessary to identify those wells in which crystals have formed such that the crystal growth conditions may be scaled up with larger quantities of the crystal material; e.g. protein. Such identification is difficult since the crystals may be very small (of the order of 1-50 microns in length). Traditionally this identification has been carried out with an optical microscope to visually identify crystalline material but information may also be gained by other techniques such as light scattering. Manual inspection is labour intensive and very slow given the large number of wells to be inspected, and re-inspected, at regular intervals. Recently, the optical identification of crystals in multi-well containers has been automated via image recognition techniques and numerous automatic or semi-automatic optical scanners are now commercially available (for example Rhombix Vision from Thermo Electron Corp., Minstrel from Rigaku Corporation, Crystal Farm from Bruker Biosciences Inc.).
Following the identification of optimal crystallisation conditions via screening, the crystal growing procedure may be scaled up with larger volumes of the material to be crystallised in the same type of multi-well containers, in the hope of growing larger crystals. In the case of proteins this may require milligrams of pure protein per well. It is then necessary to identify the crystals forming in the wells which have the highest diffraction quality. Typically this is also done using optical inspection, either manually or automatically using the same commercial products as for the crystallisation screening described above.
Finally, following the screening of crystallisation conditions, and the scaling up of the crystal growth, the most promising crystals must be harvested. This is usually done by manipulating the crystal into a cryosolution by hand and then into a cryoloop, or a capillary, such that the crystal may be mounted in an X-ray beam for observation of the diffraction quality of the crystal. Harvesting of the crystal is a particularly labour-intensive step and runs the risk that the fragile crystal will be damaged.
It is clearly of vital importance that those crystals growing successfully for the purpose of X-ray crystallography are accurately identified. As mentioned above, conventional techniques rely on optically inspecting the samples, whether manually or using an automated process. However, current optical inspection methods have a number of critical drawbacks:                Optical inspection cannot reliably identify objects growing in a liquid medium if there is little or no optical contrast.        Optical inspection cannot reliably distinguish whether objects growing in a liquid medium are crystals or amorphous material.        Optical inspection cannot reliably distinguish whether crystals growing in a liquid medium are of the material of interest or not; for example in protein crystallography, instead of protein crystals, it is very common that salt crystals may form, or in the case of protein-ligand complexes it is very common that the small molecules of the ligand may crystallise rather than the complex.        Optical inspection cannot assess the diffraction quality of a single crystal growing in a liquid medium; optical inspection can only infer the diffraction quality from the physical shape and size of the crystal, which often gives false positives, whereas the true diffraction quality can only reliably be ascertained using an X-ray beam.        
X-ray screening techniques have been suggested to overcome a number of the flaws inherent in optical screening. Preferably, these techniques will screen individual crystals in their growth environment (i.e. in situ). For example, U.S. patent application Ser. No. 10/042,929 describes an apparatus consisting of an X-ray source and an X-ray detector disposed on opposite sides of a crystal growing incubator. However, this technique is limited to ascertaining the presence or absence of diffracting material, and cannot reliably judge the diffraction quality. Furthermore, the nature of the diffraction material cannot be reliably identified using this technique. For example, the presence of salt crystals (in place of the desired protein crystals) will only be detected if the crystal is fortuitously aligned such that a Bragg condition is satisfied for one of its planes. Similar techniques are disclosed in U.S. Pat. Nos. 6,836,532 and 6,859,520 and are subject to equivalent limitations.
Moreover, X-ray screening techniques are constrained by their very nature. Inherent in any X-ray technique relying on the principles of diffraction is an inability to establish vital practical details such as the morphology of the crystal, the crystal size, and the proximity of the crystal to other objects in the droplet cannot easily be established by X-ray diffraction. Such factors are clearly essential when considering practical matters such as the ability to harvest and mount the crystal for X-ray crystallography.
The drawbacks in both optical and X-ray inspection techniques mean that very frequently the screening of crystallisation conditions can lead to false-positives, which itself leads to a costly waste of time and material attempting to scale up the growing conditions, only to find out after the object is harvested that it is not a crystal of the material of interest, or that the diffraction quality is poor. Furthermore, if the diffraction quality is shown to be poor after harvesting, it is currently not possible to know whether the harvesting process itself damaged the fragile crystal and reduced the diffraction quality.
There is consequently a need in the field to reliably identify crystals of the material of interest both in the early screening of crystallisation conditions, as well as after the growing conditions have been scaled up. There is also a need in the field to reliably assess the suitability of putative crystals for X-ray crystallography in the wider sense before they are harvested from the liquid medium in which they are grown.